ENTERIC FEVER PATHOGENS AND THEIR ANTIMICROBIAL SUSCEPTIBILITY PATTERN IN CHITWAN, NEPAL

Abstract

Enteric fever is one of the common clinical conditions in patients presenting to the hospitals. The study was carried out to assess the rate of isolation of common serotypes of enteric fever pathogens and their antimicrobial susceptibility pattern in Bharatpur, Nepal. A retrospective study was carried out in the laboratory of the Department of Microbiology, Chitwan Medical College Teaching Hospital from 15th June 2009 to 14th June 2010. A total of 4355 blood culture samples received in the laboratory were processed by standard microbiological technique to identify the causative agents and determine their antimicrobial susceptibility pattern. Isolation rate of Salmonella species was 0.9%. Among a total of 42 Salmonella isolates, 24 (57.1%) isolates were obtained as Salmonella Paratyphi A and 18 (42.9%) isolates as Salmonella Typhi. Male preponderances were seen in infections caused by both the organisms. On performing antimicrobial susceptibility by Kirby Bauer disk diffusion method, Salmonella Paratyphi A demonstrated 100% susceptibility to Amikacin, Chloramphenicol and Ofloxacin while it was least susceptible to Ampicillin (21.7%). Similarly, Salmonella Typhi was highly susceptible to Ceftriaxone (94.1%) followed by Ofloxacin (90.9%) and Cephotaxime (90%). It was also least susceptible to Ampicillin (29.4%). Multidrug resistance was found to be 16.66% among the Salmonella Typhi isolates. Enteric fever is common in Bharatpur. Salmonella Paratyphi A is the most common agent of enteric fever in this area. Both Salmonella Typhi and Salmonella Paratyphi A have developed varying frequencies of resistance against almost all commonly used antimicrobials.

Full text

Journal of Chitwan Medical College; 2012, 1(2); 26-30
Available online at: www.cmc.edu.np
A Acharya, HP Nepal, R Gautam and S Shrestha
Department of Microbiology, Chitwan Medical College, Bharatpur
Correspondence: Dr. Anju Acharya, Department of Microbiology, Chitwan Medical College (P) Ltd, Bharatpur-10, Chitwan, Nepal,
e-mail: anju_71np@yahoo.com
ENTERIC FEVER PATHOGENS AND THEIR ANTIMICROBIAL SUSCEPTIBILITY PATTERN
IN CHITWAN, NEPAL
ABSTRACT
Enteric fever is one of the common clinical conditions in patients presenting to the hospitals. The study was carried out to
assess the rate of isolation of common serotypes of enteric fever pathogens and their antimicrobial susceptibility pattern
in Bharatpur, Nepal. A retrospective study was carried out in the laboratory of the Department of Microbiology, Chitwan
Medical College Teaching Hospital from 15th June 2009 to 14th June 2010. A total of 4355 blood culture samples received
in the laboratory were processed by standard microbiological technique to identify the causative agents and determine
their antimicrobial susceptibility pattern. Isolation rate of Salmonella species was 0.9%. Among a total of 42 Salmonella
isolates, 24 (57.1%) isolates were obtained as Salmonella Paratyphi A and 18 (42.9%) isolates as Salmonella Typhi. Male
preponderances were seen in infections caused by both the organisms. On performing antimicrobial susceptibility by Kirby
Bauer disk diffusion method, Salmonella Paratyphi A demonstrated 100% susceptibility to Amikacin, Chloramphenicol
and Ooxacin while it was least susceptible to Ampicillin (21.7%). Similarly, Salmonella Typhi was highly susceptible to
Ceftriaxone (94.1%) followed by Ooxacin (90.9%) and Cephotaxime (90%). It was also least susceptible to Ampicillin
(29.4%). Multidrug resistance was found to be 16.66% among the Salmonella Typhi isolates. Enteric fever is common
in Bharatpur. Salmonella Paratyphi A is the most common agent of enteric fever in this area. Both Salmonella Typhi and
Salmonella Paratyphi A have developed varying frequencies of resistance against almost all commonly used antimicrobials.
Keywords: Antimicrobial susceptibility, Bharatpur, Enteric fever pathogens.
INTRODUCTION
Enteric fever, that is typhoid and paratyphoid fevers, is
the common name for infections caused by Salmonella
enterica serotypes Typhi and Paratyphi (commonly known as
Salmonella Typhi and Salmonella Paratyphi A respectively)1.
The infection is especially prevalent in developing countries
like Nepal with substandard water supply and sanitary
systems2. According to WHO, 16.6 million cases of enteric
fever occurs each year accounting for 600,000 deaths3
primarily in developing countries4.
Over the past decade, increasing antibiotic resistance in
Salmonella enterica has lead to a shift in the antibiotics used
against this organism from Chloramphenicol and Ampicillin
to Trimethoprim - Sulfamethoxazole, Fluoroquinolones
(Ooxacin, Ciprooxacin), and Ceftriaxone5. Even with the
use of these antibiotics, the positive response to treatment has
only been in the range of 16—40% in Nepal6.
Multidrug-resistant Salmonella Typhi (MDRST) is
epidemiologically dened as strains resistant to any two
antimicrobials in vitro even if the antimicrobials tested are
known to be clinically ineffective7. A more useful denition
of MDRST is reserved for strains resistant to all three rstline
antityphoidal antimicrobial agents, namely Ampicillin,
Chloramphenicol, and Trimethoprim- Sulphamethoxazole
(Co-trimoxazole)7.
Detection of common serotypes of enteric fever pathogens
and their antimicrobial susceptibility pattern is of utmost
importance to institute the effective therapy. After an epidemic
of enteric fever occurred in 2002 in Bharatpur where multi
drug resistant typhoid affected more than 6000 patients
in a 4 to 5 weeks period8, any evidence thereafter on the
status of isolation and susceptibility pattern of these terrible
pathogens in this particular area have not been documented.
Therefore, the present study was undertaken to assess the
rate of isolation and antimicrobial susceptibility pattern of
Salmonella species isolated from suspected cases of enteric
fever coming to Chitwan Medical College Teaching Hospital,
Bharatpur, Nepal.
MATERIAL AND METHODS
A retrospective study was carried out in the Laboratory of
Department of Microbiology at Chitwan Medical College
© 2012, JCMC. All Rights Reserved
26
ORIGINAL RESEARCH ARTICLE

© 2012, JCMC. All Rights Reserved
27
Teaching Hospital, Chitwan, Nepal. During one year period (15th June 2009 to 14th June 2010), a total of 4355 blood culture
samples were received in the Laboratory. Brain Heart Infusion (BHI) broth was used as primary culture medium for all blood culture
samples. Incubation and subcultures on blood agar and MacConkey agar were done as per the standard methods9. Suspected colonies
were further processed and identied by biochemical reactions9 and conrmed by group and type specic Salmonella antisera.
The antimicrobial susceptibility pattern of the Salmonella isolates was determined by Kirby-Bauer disc diffusion method in
compliance with CLSI guidelines10 on Muller- Hinton agar plates using Amikacin (30μg), Ampicillin (10μg), Ceftriaxone (30μg),
Cephotaxime (30μg), Chloramphenicol (30μg), Ciprooxacin (5μg), Co-trimoxazole (25μg), Gentamicin (10μg) and Ooxacin
(5μg) (Hi Media Laboratory Ltd., Mumbai, India).
RESULTS
The isolation rate of Salmonella species in this study during one year period was found to be 0.9% in total blood culture specimens.
Of 191culture positive bacterial isolates in total, 42 isolates were identied as Salmonella species. Among them, 24 isolates (57.1%)
were conrmed as Salmonella Paratyphi A and 18 isolates (42.9%) as Salmonella Typhi. Other serotypes were not isolated.
The following table (Table 1) displays the age wise distribution of the isolates. The highest number of isolates was obtained from
the age group 21-30 years and the least from the group aged more than 50 years.
Table 1: Age wise distribution of Salmonella isolates
S.N. Age group (years) Total enteric fever pathogens
Isolated S. Typhi isolates S. Paratyphi A isolates
1. Upto 10 years 4 3 1
2. 11-20 years 7 2 5
3. 21-30 years 21 8 13
4. 31-40 years 7 3 4
5. 41-50 Years - - -
6. >50 years 3 2 1
Total 42 18 24
16
14
12
10
8
6
4
2
0
No. of Isolaates
Seasons
Total Salmonella isolates
S. Typhi
S. Paratyphi
Summer Autumn Winter Spring
0
2
6
9
66
88
14
15
(-) no isolates obtained.
Male preponderances were seen in infections caused by both the organisms (male: female ratio = 2:1 for Salmonella Paratyphi A and
11: 7 for Salmonella Typhi i.e., 27:15 for all Salmonella isolates).
Figure 1: Illustrates the season wise ditribution of the Salmonella isolates. Highest number of isolates was observed in Summer
followed by Spring season and the lowest was observed in Winter season.
Figure 1: Season wise distribution of the Salmonella isolates
Acharya et al. JCMC; 2012, 1(2)

Antimicrobials Antimicrobial susceptibility pattern of Salmonella Paratyphi A
used No. of isolates tested Sensitive (%) Intermediate (%) Resistant (%)
Amikacin 17 17 (100) - -
Ampicillin 23 5 (21.7) - 18 (78.3)
Ceftriaxone 24 23 (95.8) - 1 (4.2)
Cephotaxime 20 19 (95) - 1 (5)
Chloramphenicol 20 20 (100) - -
Ciprooxacin 20 19 (95) - 1 (5)
Co-trimoxazole 19 14 (73.7) 1 (5.2) 4 (21.1)
Gentamicin 23 22 (95.7) - 1(4.3)
Ooxacin 9 9 (100) - -
Antimicrobials Antimicrobial susceptibility pattern of Salmonella Typhi
used No. of isolates tested Sensitive (%) Intermediate (%) Resistant (%)
Amikacin 13 11 (84.6) 1 (7.7) 1 (7.7)
Ampicillin 17 5 (29.4) - 12 (70.6)
Ceftriaxone 17 16 (94.1) - 1 (5.9)
Cephotaxime 10 9 (90) - 1 (10)
Chloramphenicol 15 12 (80) - 3 (20)
Ciprooxacin 16 13 (81.3) - 3 (18.7)
Co-trimoxazole 16 11 (68.8) - 5 (31.2)
Gentamicin 18 16 (88.9) - 2 (11.1)
Ooxacin 11 10 (90.9) - 1 (9.1)
Antimicrobial susceptibility pattern of the Salmonella isolates has been shown in the Tables 2 and 3. Salmonella Paratyphi A
demonstrated 100% susceptibility to Amikacin, Chloramphenicol and Ooxacin while it was least susceptible to Ampicillin (21.7%).
Similarly, Salmonella Typhi was highly susceptible to Ceftriaxone (94.1%) followed by Ooxacin (90.9%) and Cephotaxime (90%).
Least susceptibility was observed for Ampicillin (29.4%). Moreover, 3 isolates (16.66%) were found to be multidrug resistant
among the Salmonella Typhi isolates including one isolate which was resistant to all the antimicrobials tested.
Table 2: Antimicrobial susceptibility pattern of Salmonella Paratyphi A
(-) no isolates obtained
Table 3: Antimicrobial susceptibility pattern of Salmonella Typhi
(-) no isolates obtained
DISCUSSION
Enteric fever is a major health problem in developing countries attributed to poor sanitary and hygienic conditions including lack of
potable water11. The disease has remained endemic in different areas of Nepal with outbreaks occurring time and again8,12,13.
Growth positive rate of Salmonella species in our study was 0.9%. Almost similar positive rates have also been reported by Prajapati
et al13 (2.3%) from Kathmandu and Gupta et al14 (2.7%) from India. In contrast, high positive rate (23.1%) has been reported by
Amatya et al15 from Kathmandu. Relatively low positive rate observed in this study might be due to the use of antibiotics prior to
sample collection.
In contrast to previous reports2,12,13, Salmonella Paratyphi A was more commonly isolated than Salmonella Typhi in the present
study. The higher isolation rate of Salmonella Paratyphi A observed in this study indicates an increasing trend of this organism in
the recent years. Similar nding was also reported by Maskey et al16. In their observation, Salmonella Paratyphi A as a proportion of
© 2012, JCMC. All Rights Reserved
28
Acharya et al. JCMC; 2012, 1(2)

© 2012, JCMC. All Rights Reserved
29
all Salmonella isolates rose signicantly from 23.0% during
1993-1998 to 34.0% in 1999-2003. An increase in enteric
fever cases due to Salmonella Paratyphi A has also been
reported from neighboring country India 14.
Salmonella infection has been found predominantly in males
(64.3%) in this study. Previous reports13,17,18 from Nepal have
also shown higher prevalence of Salmonella infection in
males than in females. More outdoor exposure of males has
been given the possible reason for higher positive rate among
them11.
In the current study, Salmonella isolates were obtained in
the highest number from the age group 21-30 years. Our
ndings were comparable to that seen by K.C. et al19 who
had observed an average age of presentation of typhoid fever
to be 26.1 year. The particular age group seems to be more
vulnerable to exposure as they may not be having enough
care for their foods and potable drinking water due to their
busier schedule than that of other age groups.
We have observed the maximum occurrence of Salmonella
infection in the summer followed by spring. An outbreak
that had occurred in Bharatpur in 2002 was also reported to
occur in the summer8. The peak occurrence of enteric fever in
summer and rainy season has also been reported by Malla et
al12 and Sharma2, though it occurs year round in Nepal.
Drug resistance among enteric fever pathogens is considered
to be an important factor in the morbidity and mortality of the
disease. It has remained a major challenge for the clinicians as
well. The emergence of antibiotic resistant strains of bacteria
is closely linked to the irrational use of antibiotics in treating
human infections2.
Since its introduction in 1948, Chloramphenicol has been the
gold standard drug in the treatment of typhoid fever in most
parts of the world19. However, the indiscriminate use of the
drug and acquisition of plasmid mediated R factor has led to
the development of resistance of Salmonella Typhi against
the drug20. Alternative drugs suggested were Co-trimoxazole,
Ampicillin and Amoxycillin. During 1990, resistance
against Chloramphenicol, Ampicillin and Co-trimoxazole
appeared, in 50 to 52.9%21. In our study, the incidence
of Chloramphenicol resistance was found to be 20% for
Salmonella Typhi. The resistance observed by us is much
lower than the ndings of K.C. et al (63%)19. However, all the
isolates of Salmonella Paratyphi A exhibited susceptibility to
Chloramphenicol.
Very high percentage of isolates (70.6% of Salmonella
Typhi and 78.3% of Salmonella Paratyphi A) in this study
demonstrated resistance to Ampicillin. Our ndings are much
higher than the resistance observed by Khanal et al from
Dharan. It indicates that Ampicillin will have very limited
value in treating enteric fever. However, Co-trimoxazole
resistance was recorded as 31.2% for Salmonella Typhi and
21.1% for Salmonella Paratyphi A; the observation being
comparable to that of Khanal et al 22 (27%) but higher than
the nding of K.C. et al (17.4%) 19.
The increase in multi-drug resistant Salmonella is a huge
problem in developing countries2. In India 64.5% of
Salmonella Typhi was reported as being MDR in 199323.
MDR Salmonella Typhi strains were rst reported in Nepal
in 2002 during an outbreak of enteric fever in Bharatpur,
Nepal8. This outbreak of enteric fever was reported to be
a large single source outbreak due to multidrug resistant
Salmonella Typhi8. In the Present study, we have also detected
three isolates (16.66%) of MDR Salmonella Typhi including
one isolate which was resistant to all antimicrobials tested.
The nding clearly highlights a fact that the MDR isolates are
still prevalent in the Bharatpur region.
With the emergence of MDR Salmonella Typhi, Quinolone,
particularly Fluoroquinolone, has been widely used and
recommended as an alternative drug for typhoid fever
where the rst-line drug is no longer in use22. Its increasing
resistance was recorded in both Salmonella Typhi and
Salmonella Paratyphi A since the nineties.12 We have detected
5% resistance against Ciprooxacin in Salmonella Paratyphi
A and 18.7% resistance in Salmonella Typhi. The resistance
seen in Salmonella Typhi in our study is much higher than
4.3% resistance recorded by K.C. et al19. However, none of the
Salmonella Paratyphi A and only 9.1% of the Salmonella Typhi
isolates in the present studydemonstrated resistance against
Ooxacin. The salmonella isolates also exhibited relatively
lower resistance against 3rd generation Cephalosporins
(Ceftriaxone and Cephotaxime) and Aminoglycosides
(Amikacin and Gentamicin) than that against the other drugs
tested. Detection of the relatively lower resistance indicates
that these drugs along with Ooxacin may still be considered
as better options for treatment of enteric fever.
CONCLUSION
Enteric fever remains common in Bharatpur area. Occurrence
of Salmonella Paratyphi A infection is more common than that
of Salmonella Typhi infection in this region. The infections
occur throughout the year with peak incidence in summer.
These pathogens have developed varying frequencies of
resistance against almost all commonly used antimicrobials.
Detection of even multidrug resistant isolates in the present
study emphasizes on the need of continuous surveillance
on their isolation and susceptibility pattern, prudent use of
antimicrobials and institution of proper infection control
practices to limit their resistance and chances of any outbreak
that may occur in the future.
ACKNOWLEDGEMENT
We would like to thank all staffs of Laboratory of Chitwan
Medical College Teaching Hospital for their help and co-
operation to conduct this study.
Acharya et al. JCMC; 2012, 1(2)

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Acharya et al. JCMC; 2012, 1(2)