April

Indian2006
Journal of Medical Microbiology, (2006) 24 (2):101-6 101

Original Article

TREATMENT OF ENTERIC FEVER IN CHILDREN ON THE BASIS OF

CURRENT TRENDS OF ANTIMICROBIAL SUSCEPTIBILITY OF
SALMONELLA ENTERICA SEROVAR TYPHI AND PARATYPHI A
*V Manchanda, P Bhalla, M Sethi, VK Sharma

Abstract
Purpose: Recent reports indicate decreased susceptibility of S. typhi to fluoroquinolones, especially ciprofloxacin.
Chloramphenicol has been suggested as first line therapy of enteric fever in many studies. This is a prospective study
that describes the trends of antimicrobial susceptibility of S. typhi and S. paratyphi A causing bacteraemia in children and
reports therapeutic failure to ciprofloxacin and evaluates the possible use of chloramphenicol, ampicillin, ciprofloxacin
and third generation cephalosporins as first line therapy in the treatment of enteric fever in children. Methods: The
present study was conducted from April 2004 to March 2005 in a superspeciality children hospital at New Delhi. A
total of 56 S. typhi and five S. paratyphi A isolates were obtained among the 673 blood cultures performed. Antimicrobial
testing was done using disk diffusion technique (NCCLS method) for 13 antimicrobials and MICs were calculated for
ampicillin, ciprofloxacin, chloramphenicol and cefotaxime. Analysis of data was done using WHONET software. Results:
All 56 isolates of S. typhi were sensitive to amoxycillin+clavulanate, gentamicin, cefixime, cefotaxime and ceftazidime.
Multidrug resistance (MDR, resistance to three drugs) was seen in 22 cases (39%) and resistance to five drugs was seen
in 12 cases (21%). Only two isolates were resistant to chloramphenicol (3%). MIC90 for ampicillin, chloramphenicol,
ciprofloxacin and cefotaxime were 1.0 µg/ml, 4.0 µg/ml, 64 µg/ml and 0.125 µg/ml respectively. All S. paratyphi A isolates
were sensitive to ampicillin and chloramphenicol and resistant to nalidixic acid.MIC distribution data for chloramphenicol
revealed elevated MIC but still in susceptible range. Conclusions: There is an urgent need for further clinical studies to
evaluate response to chloramphenicol in such cases. Antimicrobial susceptibility data and MIC distribution favour use of
ampicillin as a drug of choice for the treatment of enteric fever. Third generation cephalosporins are also useful but their
use should be restricted for complicated cases.

Key words: Enteric fever, Salmonella, chloramphenicol, fluoroquinolones

Multidrug resistant (MDR) strains (resistant to
chloramphenicol, ampicillin and cotrimoxazole) of Salmonella
enterica serovar Typhi (S. typhi) are increasingly being
reported from India and worldwide. 1-4 Third-generation
cephalosporins and fluoroquinolones have been found
effective in treatment of these cases.5,6 However, isolates of
S. typhi with reduced susceptibility to fluoroquinolones (as
indicated in the laboratory by resistance to nalidixic acid) have
now appeared in the Indian subcontinent and other regions.7-
12
These nalidixic acid resistant but ciprofloxacin sensitive
strains have increased minimum inhibitory concentrations
(MICs) for ciprofloxacin, although they are still within the
current NCCLS range for susceptibility (0.12-0.5 µg/ml).13
Therapeutic failures to ciprofloxacin have been reported in
these cases of typhoid fever.13-15 Furthermore, an isolate of S.
typhi from Bangladesh with high-level resistance to ceftriaxone
*Corresponding author (email: <manchandavikas@hotmail.com>)
Department of Clinical Microbiology (VM), Department of Pediatrics
(MS), Department of Microbiology (PB, VKS); Maulana Azad
Medical College and Associated Chacha Nehru Children Hospital,
Geeta Colony, New Delhi - 110 031, India.
Received : 31-05-05
Accepted : 07-10-05
has already been reported. 16 However, reemergence of
chloramphenicol susceptible strains have also been reported
during recent years.17,18
A prospective study was planned to study the extended
patterns of antimicrobial susceptibilities of S. typhi and S.
paratyphi A isolated from the blood cultures at our hospital.
Additionally, clinical therapeutic responses were observed for
few of these cases to ciprofloxacin, ampicillin and cefixime.
Also, the possible use of chloramphenicol, ampicillin,
ciprofloxacin and third generation cephalosporins as first line
therapy in the treatment of enteric fever in children was
evaluated.
Materials and Methods
The study was conducted at Chacha Nehru
Superspeciality Children Hospital, Delhi from May 2004 to
April 2005. Blood samples of 673 children with suspected
enteric fever were subjected to blood culture. Salmonella
enterica serovar Typhi was isolated in 56 cases (8.3%) and
Salmonella enterica serovar Paratyphi A in five (0.7%) cases.
Antimicrobial susceptibility patterns were determined using
following commercial antimicrobial disks (HiMedia, India):
chloramphenicol (30 µg), nalidixic acid (30 µg), ampicillin (10

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102 Indian Journal of Medical Microbiology vol. 24, No. 2

µg), azithromycin (15 µg), cotrimoxazole (1.25/23.75 µg),
ofloxacin (5 µg), ciprofloxacin (5 µg), tetracycline (30 µg),
cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg)
amoxycillin + clavulanic acid (20/10 µg) and gentamicin (10 µg).
Antimicrobial susceptibility testing was performed in
accordance with the National Committee for Clinical Laboratory
Standards (NCCLS) methodology.19
Minimum inhibitory concentrations (MICs) for
ciprofloxacin and cefotaxime were determined using E - test
(AB Biodisk, Solana, Sweden). Broth microdilution testing was
performed to determine MICs for chloramphenicol and
ampicillin in accordance with the National Committee for
Clinical Laboratory Standards (NCCLS). 20 The reagent
powders were dissolved in Mueller-Hinton broth and
distributed to the wells of microdilution trays. Each tray was
inoculated with 5×104 CFU per well to yield a final volume of
0.1 ml per well. Final dilutions ranged from 256 µg/mL - 0.25
µg/mL. The trays were incubated overnight at 35°C. Quality
control was monitored by using Escherichia coli ATCC 25922.
The recorded MIC of each antibiotic was the lowest
concentration that completely inhibited visible growth of the
test strain. The MIC at which 50% of the isolates tested were
inhibited (MIC50) and MIC90 were calculated in accordance
with the current NCCLS methodology and using WHONET
software ver 5.1.
Results
All 56 isolates of S. typhi were found sensitive to
amoxycillin + clavulanate combination, gentamicin, cefixime,
cefotaxime and ceftazidime. Details of antimicrobial
susceptibility patterns are shown in Table 1. Seven S. typhi
isolates were sensitive to all drugs tested. Two isolates showed
resistance to chloramphenicol. Multi-drug resistance (MDR,
resistant to ≥ 3 drugs) was observed in 22 cases (39%) and
resistance to ≥ 5 drugs was seen in 12 cases (21%). All five S.
paratyphi A isolates were sensitive to ampicillin and
chloramphenicol and resistant to nalidixic acid.
Fourteen different types of antimicrobial profiles were
observed for S. typhi. Nineteen isolates were resistant only
to nalidixic acid (N), three each were additionally resistant to
azithromycin (NZ) and tetracycline (NT). Eight isolates were
found resistant to nalidixic acid, cotrimoxazole (Co), ofloxacin
(O), ciprofloxacin (P) and tetracycline (T) (NCoOPT). Detailed
antimicrobial resistance profiles are summarized in Table 2. Five
S. paratyphi A isolates had three different resistance profiles.
Three isolates were resistant to nalidixic acid and azithromycin
(NZ) and one isolate each was resistant to nalidixic acid alone
(N) and to nalidixic acid and tetracycline (NT). Seven (13%)
strains were sensitive to all drugs.
Minimum inhibitory concentrations (MICs) range and their
distribution among the isolates are shown in Table 1 and
Figures 1a-1d.
Nalidixic acid resistant S. typhi (NARST) isolates were
obtained in 48 cases and nalidixic acid sensitive isolates were
found among eight cases of suspected typhoid fever. Among
NARST, only 12 isolates met the criteria of current NCCLS
break points for ciprofloxacin resistance and only two isolates
had MIC < 0.12 µg/mL. The remaining 42 isolates had
increased MICs between ≥ 0.12 - <1 µg/mL (Fig. 2a and 2b).
Scatter plot (Fig. 2a) compares nalidixic acid zone diameters
and ciprofloxacin MICs. Large numbers of isolates were found
in the area of nalidixic acid resistance with slightly increased
MICs for ciprofloxacin (0.12 -1 µg/mL), but still below the
current break point (<1 µg/mL). Another scatter plot (Fig. 2b]
compares nalidixic acid zone diameters with that of
ciprofloxacin zone diameters. In this figure, majority of the
isolates fall in nalidixic acid resistant but ciprofloxacin
sensitive area. Among S. paratyphi A isolates, MIC for

Table 1: Antimicrobial susceptibility patterns of S. typhi

Antimicrobials R I S MIC range MIC50 MIC90 Geometric mean
n (%) n (%) n (%) (µg/mL) (µg/mL) (µg/mL) MIC (µg/mL)
Chloramphenicol 2 (3.6) 1 (1.8) 53 (94.6) 64 - 0.5 1 2 1.08
Nalidixic acid 48 (85.7) 0 8 (14.3) ND ND ND ND
Ampicillin 0 1 (1.8) 55 (98.2) 128 - 0.125 0.125 1 0.237
Azithromycin 0 14 (25) 42 (75) ND ND ND ND
Cotrimoxazole 17 (30.4) 0 39 (69.6) ND ND ND ND
Ofloxacin 11 (19.6) 0 45 (80.4) ND ND ND ND
Ciprofloxacin 12 (21.4) 2 (3.6) 42 (75) >32 - 0.012 0.25 64 0.386
Tetracycline 19 (33.9) 5 (8.9) 32 (57.1) ND ND ND ND
Cefixime 0 0 56 (100) ND ND ND ND
Cefotaxime 0 0 56 (100) 0.125 - 0.023 0.064 1.25 0.073
Ceftazidime 0 0 56 (100) ND ND ND ND
Amoxycillin + Clavulanate 0 0 56 (100) ND ND ND ND
Gentamicin 0 0 56 (100) ND ND ND ND
S- Sensitive; I- Intermediate; R-Resistant; MIC - Minimum inhibitory concentration; ND -Not determined

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April 2006 Manchanda et al – Treatment of Enteric Fever in Children 103

Figure 1a: MIC distribution of ciprofloxacin in S. typhi isolates. R, Figure 1b: MIC distribution of chloramphenicol in S. typhi isolates.
Resistant; I, Intermediate resistant; S, Sensitive Lines in plot area denote boundaries for resistance and susceptibility
cut off points. R, Resistant; I, Intermediate resistant; S, Sensitive.

Figure 1c: MIC distribution of ampicillin in S. typh i isolates. Lines in

plot area denote boundaries for resistance and susceptibility cut off Figure 1d: MIC distribution representative of third generation
points. R, Resistant; I, Intermediate resistant; S, Sensitive. cephalosporins (cefotaxime in this figure) in S. typhi isolates. Lines in
plot area denote boundaries for resistance and susceptibility cut off
Table 2: Antimicrobial resistance profiles for Salmonella points. R, Resistant; I, Intermediate resistant; S, Sensitive.
typhi
Profile N (%) ciprofloxacin ranged between 0.125-0.75 µg/mL and for
T 1 (2)
chloramphenicol ranged between 0.5-8 µg/mL. MIC
N 19 (34)
distribution for cefotaxime was between 0.064-0.094 µg/mL. All
NT 3 (5)
five isolates had MIC of 1.0 µg/ml for ampicillin.
NZ 3 (5)
Oral ciprofloxacin (10 mg/kg/day) was prescribed to the first
NCoT 1 (2)
five patients infected with nalidixic acid resistant but
NZT 4 (7)
ciprofloxacin sensitive S. typhi (MIC > 0.64 µg/mL but < 1 µg/
NZP 1 (2)
mL) and were reviewed after five days. Of these five patients,
CNP 1 (2)
only one responded to the therapy. In the remaining four
NCoPT 1 (2)
patients, higher doses of ciprofloxacin (20 mg/kg/day) were
NZCoT 2 (4)
advised and they were reviewed after another three days. Only
CNCoT 1 (2)
two of them responded by this time. Oral cefixime (16 mg/kg/
NCoOPT 8 (14)
day) was advised in the other two and an excellent response
CNAZCo 1 (2)
was observed. During the rest of the study none of the culture
NZCoOPT 3 (5)
positive patients of typhoid fever were advised ciprofloxacin.
C- Chloramphenicol, N- Nalidixic acid, A- Ampicillin, Z-
Azithromycin, Co- Cotrimoxazole, O- Ofloxacin, P- Ciprofloxacin, They were advised either oral amoxycillin (100 mg/kg/day)
T- Tetracycline or cefixime (20 mg/kg/day) to which, they responded well. The

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104 Indian Journal of Medical Microbiology
Figure 2a: Scatterplots for nalidixic acid (disk diffusion) versus
ciprofloxacin (MICs) for Salmonella enterica serovar Typhi. Lines in
plot area denote boundaries for resistance and susceptibility cut off
points. Numbers in plot area denotes number of isolates. R, Resistant;
I, Intermediate resistant; S, Sensitive.
follow up was uneventful.
Discussion
In the preantibiotic era, typhoid fever case fatality rates
approached 20%. Treatment with effective antimicrobial agents
- ampicillin, chloramphenicol, cotrimoxazole and later
ciprofloxacin - has reduced the case fatality rate to less than
1%. 21 The resistance pattern for S. typhi varies with
geographical locations.
Many studies have suggested that patients in Indian
subcontinent or with the history of travel to the Indian
subcontinent should receive ciprofloxacin as first line
therapy.8,22 However, S. typhi isolates resistant to ciprofloxacin
and ceftriaxone (MIC, 64 mg/L) have been reported.13,16,23-27 S.
typhi strains with reduced susceptibility to fluoroquinolones
have become a major problem in Asia and other parts of the
world.12 Although they were reported to be susceptible to
fluoroquinolones, by disk testing with the use of
recommended break points, these organisms were resistant to
nalidixic acid and the MIC of fluoroquinolones for these
strains was 10 times higher than that for fully susceptible
strains. This reduction in susceptibility results in a poor
clinical response to treatment. There is unpredictable response
to treatment with ciprofloxacin in patients infected with these
kinds of S. typhi strains. Selective pressure on the bacterial
population by uncontrolled use of quinolones has likely led
to emergence of resistance to this group of antimicrobials.
This has been attributed to point mutation in quinolone
resistance determining region (QRDR) of the topoisomerase
gene gyr A, characteristically occurring at position 83 of the
DNA gyrase enzyme (changing serine to phenylalanine) and
position 87 (changing aspartate to tyrosine or glycine).9
However, other mechanisms of resistance such as decreased
permeability and active efflux of the antimicrobial agent may
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vol. 24, No. 2
Figure 2b: Scatterplots for nalidixic acid (disk diffusion) versus
ciprofloxacin (disk diffusion) for Salmonella enterica serovar Typhi.
Lines in plot area denote boundaries for resistance and susceptibility
cut off points. Numbers in plot area denotes number of isolates. R,
Resistant; I, Intermediate resistant; S, Sensitive.
be involved. In other Enterobacteriaceae, higher levels of
quinolone resistance have been associated with additional
mutations in the gyr A gene, mutations in other topoisomerase
genes, or alterations in fluoroquinolone uptake.13 No such
mutations have been reported yet in S. typhi, although there
are sporadic reports of completely fluoroquinolone resistant
isolates.
Because clinical response to fluoroquinolones in patients
infected with nalidixic acid resistant strains is greatly inferior
to the response in those infected with nalidixic acid-susceptible
strains, the break points for the classification of S.typhi strains
according to their susceptibility to fluoroquinolones should
be reviewed. A practical approach would be to classify strains
that are resistant to nalidixic acid but susceptible to
fluoroquinolones according to current disk testing criteria as
resistant to quinolones or non susceptible to fluoroquinolones
as suggested by other authors as well.12 All strains that have
intermediate susceptibility or resistance to fluoroquinolones
on disk testing (as defined by NCCLS guidelines) should be
considered fluoroquinolone resistant. There are reports that
show that treatment of patients infected with similar isolates
is at potential risk of therapeutic failure.13
This study showed that resistance to nalidixic acid is
generally associated with increased MICs of ciprofloxacin.
Hence, nalidixic acid susceptibility testing must be included
with ciprofloxacin susceptibility testing in routine
microbiological laboratory. Treatment with ciprofloxacin must
be avoided in nalidixic acid resistant isolates of S. typhi
although higher doses may be helpful in a few cases.
Most of the isolates in this study were found susceptible
to chloramphenicol and ampicillin. Percentage resistance,
intermediate resistance and sensitive (%RIS) data in Table 1
show that ampicillin and chloramphenicol should be
April 2006 Manchanda et al – Treatment of Enteric Fever in Children 105
considered for the treatment of typhoid fever in children in
Delhi, however, MIC distribution data projects different
scenario. As discussed above, infection with isolates with
raised MICs but still in susceptible range for ciprofloxacin (Fig.
1a) has a potential risk of therapeutic failure. MIC distribution
of chloramphenicol closely resembles that of ciprofloxacin in
raised MICs but still in susceptible range (Fig. 1b). Hence,
theoretically, treatment with chloramphenicol also has a
potential risk of therapeutic failure. More studies are needed
to evaluate reuse of chloramphenicol as the first choice for
treatment of typhoid fever. Minimum inhibitory concentrations
of ampicillin and cefixime on the other hand were well below
the susceptible limits and hence may be used safely in the
treatment of typhoid fever.
To conclude, MIC distribution for ciprofloxacin and
chloramphenicol had striking similarity, i.e, raised MICs but
still in susceptible range. However, MICs distribution of
ampicillin and cefotaxime was well below the susceptible limits.
Therapeutic failures for ciprofloxacin have been observed.
Similar responses may be observed for chloramphenicol.
Therapeutic choice for typhoid fever should be carefully
selected. Ampicillin and probably not chloramphenicol may be
reconsidered as first line of therapy in uncomplicated cases
of typhoid fever in children.
References
1. Ackers ML, Puhr ND, Tauxe RV, Mintz ED. Laboratory-based
surveillance of Salmonella serotype Typhi infections in the
United States: antimicrobial resistance on the rise. JAMA
2000;283:2668-773.
2. Ahuja S, Mago ML, Singh H, Saxena SN. Multiple drug
resistance pattern of Salmonella strains in India during 1976.
Indian J Med Res 1979;70:702-9.
3. Madhulika U, Harish BN, Parija SC. Current pattern in
antimicrobial susceptibility of Salmonella Typhi isolates in
Pondicherry. Indian J Med Res 2004;120:111-4.
4. Vaze S, Sharma KB. In vitro sensitivity of S. typhi and S.
paratyphi A organisms against furazolidone, ampicillin and
chloramphenicol. Indian J Med Sci 1971;25:859-61.
5. Cao XT, Kneen R, Nguyen TA, Truong DL, White NJ, Parry
CM. A comparative study of ofloxacin and cefixime for
treatment of typhoid fever in children. The Dong Nai Pediatric
Center Typhoid Study Group. Pediatr Infect Dis J 1999;18:245­
8.
6. White NJ, Parry CM. The treatment of typhoid fever. Curr
Opin Infect Dis 1996;9:298-302.
7. Wain J, Hoa NT, Chinh NT, Vinh H, Everett MJ, Diep TS, et
al. Quinolone-resistant Salmonella typhi in Vietnam: molecular
basis of resistance and clinical response to treatment. Clin Infect
Dis 1997;25:1404-10.
8. Rowe B, Ward LR, Threlfall EJ. Ciprofloxacin-resistant
Salmonella typhi in the UK. Lancet 1995;346:1302.
www.ijmm.org
9. Brown JC, Shanahan PM, Jesudason MV, Thomson CJ, Amyes
SG. Mutations responsible for reduced susceptibility to 4­
quinolones in clinical isolates of multi-resistant Salmonella typhi
in India. J Antimicrob Chemother 1996;37:891-900.
10. Jesudason MV, Malathy B, John TJ. Trend of increasing levels
of minimum inhibitory concentration of ciprofloxacin to
Salmonella typhi. Indian J Med Res 1996;103:247-9.
11. Chitnis V, Chitnis D, Verma S, Hemvani N. Multidrug-resistant
Salmonella typhi in India. Lancet 1999;354:514-5.
12. Kapil A, Renuka, Das B. Nalidixic acid susceptibility test to
screen ciprofloxacin resistance in Salmonella typhi. Indian J Med
Res 2002;115:49-54.
13. Crump JA, Barrett TJ, Nelson JT, Angulo FJ. Reevaluating
fluoroquinolone breakpoints for Salmonella enterica serotype
Typhi and for non-Typhi salmonellae. Clin Infect Dis
2003;37:75-81.
14. Threlfall EJ, Ward LR, Skinner JA, Smith HR, Lacey S.
Ciprofloxacin-resistant Salmonella typhi and treatment failure.
Lancet 1999;353:1590-1
15. Nguyen TC, Solomon T, Mai XT, Nguyen TL, Nguyen TT,
Wain J, et al. Short courses of ofloxacin for the treatment of
enteric fever. Trans R Soc Trop Med Hyg 1997;91:347-9.
16. Saha SK, Talukder SY, Islam M, Saha S. A highly ceftriaxone­
resistant Salmonella typhi in Bangladesh. Pediatr Infect Dis J
1999;18:387.
17. Mandal S, Mandal MD, Pal NK. Reduced minimum inhibitory
concentration for Salmonella enterica serovar Typhi. Indian J
Med Sci 2004;58:16-23.
18. Chandel DS, Chaudhry R, Dewan B, Pandey A, Dey AB. Drug-
Resistant Salmonella enterica Serotype Paratyphi A in India.
Emerg Infect Dis 2000;6:420-1.
19. National Committee for Clinical Laboratory Standards.
Performance standards for antimicrobial susceptibility testing.
Ninth informational supplement, M100-S10. National
Committee for Clinical Laboratory Standards: Wayne,
Pennsylvania; 2000.
20. National Committee for Clinical Laboratory Standards. Method
for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically. Approved standard M7-A5. National
Committee for Clinical Laboratory Standards: Wayne, PA; 2000.
21. Mermin JH, Townes JM, Gerber M, Dolan N, Mintz ED, Tauxe
RV. Typhoid fever in the United States, 1985-1994: Changing
risks of international travel and increasing antimicrobial
resistance. Arch Intern Med 1998;158:633-8.
22. Gulati S, Marwaha RK, Prakash D, Ayyagari A, Singhi S, Kumar
L, et al. Multi-drug-resistant Salmonella typhi—a need for
therapeutic reappraisal. Ann Trop Paediatr 1992;12:137-41.
23. Atkins BL, Gottlieb T. Emerging drug resistance and vaccination
for typhoid fever. JAMA 1998;279:579-80.
24. Chandra R, Srinivasan S, Nalini P, Rao RS. Multidrug resistant
106 Indian Journal of Medical Microbiology vol. 24, No. 2

enteric fever. J Trop Med Hyg 1992;95:284-7. multidrug resistant enteric fever—changing pattern and emerging
resistance to ciprofloxacin. J Assoc Physic India 1994;42:599­
25. Rowe B, Ward LR, Threlfall EJ. Multidrug-resistant Salmonella 600.
typhi: a worldwide epidemic. Clin Infect Dis 1997;24:S106-9.
27. Bhatia RS. Typhoid fever, not responding to ciprofloxacin
26. Daga MK, Sarin K, Sarkar R. A study of culture positive therapy. J Assoc Physic India 1992;40:705-6.

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