Salmonella typhi has long been recognized to cause typhoid fever in
various parts of the world. Although the incidence of typhoid fever is falling
in developed country, the disease is still endemic in many parts of the world
including Bangladesh (Hoque et al., 1992; Bhattacharya
and Das, 2000; Coovadia et al., 1992; Gautam
et al., 2002). Typhoid fever is usually associated with bacteremia
and inflammatory destruction of intestine and other organs and is fatal to adults,
children and immunocompromised persons (Afroj et al.,
2011; Islam et al., 2007).
Emergence of multi-antibiotic resistant Salmonella spp., especially
fluoroquinolones and third-generation cephalosporin resistant Salmonella
spp., has been reported worldwide and is considered as a serious problem due
to limitations of the effective treatment of human infections (Albert
et al., 1991; Saha and Saha, 1994). Therefore,
now it is a prime concern to consider new, effective and alternative choice
of drug in forthcoming days to combat against typhoid caused by S. enterica
serovar Typhi and Paratyphi resistant to traditional antibiotics like ciprofloxacin
(Jesudason et al., 1996; Rahman
et al., 2005).
Appearance of Multi drug resistance Salmonella typhi resulted in a pressing
need to test newer antimicrobials, develop their dose regimen for the treatment
of typhoid fever (Khan and Hoque, 1992). And to find
the mechanism of acquiring resistance in these multidrug resistance Salmonella
typhi isolates (Akter et al., 2012). Knowledge
of the prevalent species of Salmonella in a country and their antibiotic
susceptibility pattern is therefore important to determine any preventive strategy
(Shahriar and Kabir, 2010).
The present study aimed to assess prevalence of bloodstream Salmonella
infections in patients with typhoid fever and to determine the antibiotic susceptibility
patterns of the isolates.
MATERIALS AND METHODS
Duration and place of study: This study was conducted on 656 samples
of blood collected from same number of patients over a period of one year (January
to December, 2012). Blood samples were collected from patients suspected of
suffering from septicemia and typhoid fever admitted to Dhaka Medical College
Hospital. The age of patients included in this study ranged from 02 to 61 years.
The population under study included both male and female patients. With the
permission of the hospital authority and institutional ethical review committee,
informed consent was obtained from each of the participating subjects.
Collection of blood samples: The skin of venipuncture site of the patient
was first cleaned with 95% alcohol. Ten ml of blood was drawn into a 10 mL disposable
pyrogen free syringe. Five milliliter of blood was inoculated into blood culture
bottle containing 45 mL of brain-heart infusion broth. These bottles were immediately
transported to Laboratory (Saha et al., 1999).
Isolation of Salmonella typhi and Salmonella paratyphi:
Blood cultures bottles were incubated aerobically at 37°C for one week.
These bottles were examined daily throughout the week of incubation. When growth
appeared on any of the medium, a gram stain film was made. If gram negative
bacilli were detected, the culture from blood culture bottle was inoculated
onto a blood agar, XLD agar and MacConkeys agar plate (Saha
and Saha, 1994).
Biochemical identification of Salmonella typhi: The API 20E was
used for biomedical identification of Salmonella typhi and Salmonella
paratyphi. The system consists of a plastic strip with 20 microtubes containing
dehydrated substrate of ortho-nitrophenyl-galactopyranoside. The incubation
box was prepared by putting 5 mL of sterile water into the honey combed wells
of the tray. This was done to create a humid chamber. The strip was then place
in the tray. A single isolated colony of suspected Salmonella was picked
up from MacConkeys plate. It
was emulsified in sterile saline to achieve homogenous bacterial suspension.
With a sterile pipette, the tubes and cupules of citrate, VP and gelatin were
filled with bacterial suspension. The remaining tubes and cupules were filled
with bacterial suspension. Anaerobic environment was created in the tests ADH,
LDC, ODC, URE and H2S by overlaying mineral oil. The box was closed
and incubated at 37°C for 24 h. A MacConkey,s agar plate was
inoculated with the same homogenous bacterial suspension to check the purity
of the bacterial suspension and incubated at 37°C for 24 h.
Serological identification of Salmonella typhi and Salmonella
paratyphi: Anti-Salmonella agglutinating sera contain Salmonella
typhi, Salmonella paratyphi A, B, C and Vi from BioMerieux Laboratory
was used. Kauffmann and White Scheme was followed for serological confirmation
of Salmonella typhi and Salmonella paratyphi. One drop of agglutinating
sera was placed on a clean glass slide. One colony of the test strain of was
picked up with a loop from MacConkey,s agar plate. This bacterial
culture and agglutinating serum was mixed slowly with a sterile stick. When
fully mixed the slide was rotated for 5-10 sec. The agglutination was watched
by naked eye. Positive and negative controls were tested in the similar way
on the same slide (Morshed et al., 1986).
Antibiotic susceptibility testing: Twenty two strains were tested for
antibiotic resistance by the standard agar disc diffusion technique (Bauer
et al., 1966) on Mueller Hinton agar using commercial discs (Oxoid,
UK).The following antibiotics with the disc strength in parentheses were used:
Ciprofloxacin (Cip, 5 μg), Cephotaxime (Cep, 30 μg), Ceftriaxone (Cef,
30 μg), Cotrimoxazoll (Cot, 25 μg), Ampicillin (Amp, 10 μg),
Erythromycin (Ery, 15 μg) and Nalidixic acid (Nal, 30 μg). A control
strain of E. coli ATCC 25922 was included in each plate. Antimicrobial
breakpoints and interpretation were taken from the CLSI standards (Clinical
and Laboratory Standards Institute, 2006), formerly NCCLS).
Isolation of Salmonella spp.: A total of 153 Salmonella
were isolated from the blood samples. Isolation was based on the colony characteristics
on Mac Conkey agar and XLD agar. They produced colorless colonies on MacConkey
agar as they does not ferment lactose and black centered red colonies on XLD
Biochemical and serological identification of Salmonella typhi and
Salmonella paratyphi: API 20E was used for biochemical identification
and differentiation of Salmonella typhi and Salmonella paratyphi.
All the isolates showed negative results in ONPG, ADH, Citrate, urease, tryptophane
deaminase, Indole, Voges proscauer, Gelatine, inositol, saccharose and amylose
test and showed positive results in LDC, ODC, H2S, Glucose, mannose,
sorbose, rhaffinose, mellibiose and arabinose test. Upon serology, 94 isolates
agglutinates with Salmonella typhi polyvalent antisera and 59 isolates
agglutinates with Salmonella paratyphi polyvalent antisera, confirming
Rate of Salmonella typhi and Salmonella paratyphi isolated
from blood cultures: The month wise percent prevalence of Salmonella
spp. (Salmonella typhi and Salmonella paratyphi) isolated from
blood cultures were as follows,- In January it was 40%, February 25%, March
27%, April 24%, May 25%, June 19%, July 28 %, August 20%, September 17%, October
15% November 23% and December 35% (Fig. 1). The typhoid and
para typhoid fever was prevalent throughout the year. Average prevalence rate
of Salmonella in blood was 24.8%. Peak prevalence was in the month of
January 2012, it was about 40%. The results show that a rise in typhoid fever
was observed during the months of June to August, This was expected because
of the rains in the country, un-hygienic conditions prevailed and this resulted
in the increase in the percentage of typhoid fever.
Month wise prevalence of Salmonella typhi and Salmonella paratyphi
is shown in Fig. 2. Highest number of Salmonella was
isolated in July, 2012 of which 20 were Salmonella typhi and 9 were Salmonella
Age distribution of bloodstream Salmonella positive patients showed
that young child and neonates and elderly persons are more susceptible to Salmonella
infection in blood. It is likely that their immune system is weaker than healthy
adults and thus they are prone to septicemia.
|| Month wise distribution pattern of Salmonella spp.
||Month wise prevalence of Salmonella typhi and Salmonella
||Age distribution of bloodstream Salmonella positive
Patients of 16-45 years are less susceptible to Salmonella infections
Sex distribution of Salmonella infected patients indicate that males
are more susceptible to infection than female. Of 153 Salmonella positive
patients, 91 were male and 62 were female. Of 94 Salmonella typhi infected
patient, 59 were male and 35 were female and of 59 Salmonella paratyphi
infected patietns, 32 were male and 27 were female (Fig. 4).
Sensitivity/resistance of Salmonella isolates to antimicrobial compounds:
All the 94 isolates of Salmonella typhi and 59 isolate of Salmonella
paratyphi were tested against nine antimicrobial agents, namely Amoxicillin,
Chloramphenicol, Trimethoprim-sulfamethoxazole, Cefotaxime. Cefipime, Ceftriaxone,
Nalidixic acid, Ciprofloxacin and Meropenem. All the mentioned drugs are the
drug of choice for the treatment of typhoid fever.
|| Sex distribution of Salmonella positive patients
||Antibiotic resistance pattern of isolated Salmonella typhi
and Salmonella paratyphi
Among the 94 Salmonella typhi isolate, more than 90% isolates were resistant
to cefixime, ceftriaxone, meropenem and cefipime- all of which are third generation
cephalosporin antibiotic. Resistance to ciprofloxacin was 82% and to chloramphenicol
was 70% and to Trimethoprim-sulfamethoxazole was 68%. Only Nalidixic acid was
found to be effective as only 20% of the Salmonella typhi isolates were
resistant to nalidixic acid. Among 59 Salmonella paratyphi isolate, more
than 85% isolates were resistant to cefixime, ceftriaxone, cefipime, ciprofloxacin
and Meropenem. Moderate resistance was found against amoxicillin (44%) and Trimethoprim-Sulfamethoxazole
(31%). Nalidixic acid was found most effective against Salmonella paratyphi.
Antibiotic resistance pattern of Salmonella typhi and Salmonella paratyhpi
is shown in Fig. 5.
Typhoid fever caused by Salmonella typhi and Salmonella paratyphi
is one of the most common infections in Bangladesh (Rahman
et al., 2002). Strains of Salmonella typhi and Salmonella
paratyphi resistant to commonly used antibiotics such as chloramphenicol,
Amoxicillin, Trimethprim-sulfamethoxazole, Ciprofloxacin are emerging in many
parts of the world including Bangladesh (Alam et al.,
2010). Therefore, isolation of Salmonella from typhoid patient samples such
as blood and assessment of antibiotic susceptibility pattern of those isolates
is necessary to select drug of choice to treat such infections (Datta
et al., 1981).
This study was carried out to have an idea of the antibiotic susceptibility
pattern of Salmonella spp. (Salmonella typhi and Salmonella
paratyphi) isolated from typhoid patients
blood. Blood samples were collected from patients admitted with typhoid fever
during January to December, 2012. A total of 656 blood samples have been collected.
Out of 656 samples of blood cultures 153 turned out to be positive for Salmonella
spp. which is about 23% of the total samples. Though most of the patients have
symptoms for typhoid, the reason for the low Salmonella positive blood samples
may be due to antibiotic medication during blood collection.
Salmonella spp. isolated from patients were identified by biochemical
tests using API20E reaction system and all the isolates produced typical reactions
of Salmonella. The isolates were further confirmed and identified to serotype
level by serotyping with polyvalent antisera against S. typhi and S.
paratyphi. Salmonella typhi (94) has been found to be more prevalent
than S. paratyphi (59). Salmonella infection in blood was registered
throughout the year but highest prevalence was found in December-January.
Neonates and young (age 2-15) and elderly persons (age 46-60) were more susceptible
to Salmonella infection may be due to their weaker immune system. Healthy
adults are less susceptible to bloodstream infection of Slamonella. Males were
more infected than females and the same pattern was observed in case of Salmonella
spp., Salmonella typhi and Salmonella paratyphi infection.
A high percentage of the isolates showed resistance to first line (amoxicillin,
chloramphenicol, Trimethoprim-Sulfamethoxazole) and second line drugs (cefotaxime,
cefipime, ceftriaxone, ciprofloxacin). Only Nalidixic acid showed to be effective
against these isolates.
Several previous studies reported bloodstream Salmonella spp. in patients
from Bangladesh. In a study by Alam et al. (2010),
66% patients were Salmonella spp. positive and one third of the isolated
Salmonella typhi were multi drug resistance. The isolates were most resistant
to amoxicillin, cotrimoxazole and chloramphenicol.
In another study by Shadia et al. (2011), among
385 isolated Salmonella sp., 304 (79%) were Salmonella enterica serovar
Typhi and 81 (21%) were Salmonella enterica serover Paratyphi. About
40% of the Salmonella typhi isolates were resistant to ampicillin, chloramphenicol
and co-trimoxazole compared to only 1.8% S. paratyphi. All S. typhi
and Paratyphi A were sensitive to ceftriaxone.
Chowdhury and Anwar (2010) isolated 12 MDR Salmonella
isolates from hospital waste those were resistant to ciprofloxacin, ampicillin,
amoxicillin and penicillin. Presence of such MDR Salmonella in hospital waste
indicates bloodstream infection of patients with Salmonella. In another
study, 943 Salmonella typhi were isolated from patients with typhoid
fever and more than 50% isolates were resistant to ampicillin, cotrimoxazole
and chloramphenicol and more than 90% isolates were resistant to nalidixic acid
though resistance to ciprofloxacin was very low (Mahmud
et al., 2010). Similar results were also found in the study of Sarker
et al. (2010) where resistance was higher to ampicillin, cotrimoxazol
and chloramphenicol and all isolates were sensitive to ceftriaxon and ceftazidim.
Our study reports lower prevalence rate than the above mentioned studies, may
be due to lower sample size. But isolates of the present study pose higher resistance
compared to the reported cases from Bangladesh.
This study contradicts with the study of Mahmud et
al. (2010) in terms of sensitivity of Salmonella typhi and S.
paratyphi to nalidixic acid as our isolates were sensitive to nalidixic
acid compared to high resistance reported by Mahmud et
This study is in conjunction of the previous studied warns us about increasing
prevalence of bloodstream Salmonella infection in a wide group of patients and
their increased resistance to the first line and second line drugs of choice.
Overuse and availability of antimicrobial is a commonpactice in this part of
the world. The selective pressure of un-restricted antimicrobial usage has probably
contributed to the genesis of resistant Salmonella typhi (Mills-Robertson
et al., 2002). In Dhaka antimicrobial are available from chemist
shops without legal prescriptions. This encourages the patients to buy antimicrobials
from the counter and use them without consultation with the doctor. By doing
so, these patients often wrong antimicrobial drug which help in selection of
resistance in bacteria rather than curing the patient (Saha
and Saha, 1994). This self-medication is often taken for wrong duration
which further helps in selection of resistant bacteria against these antimicrobial.
Very often antimicrobials are prescribed without determining the causative organism
or its susceptibility to antimicrobial. When antimicrobials are prescribed for
infections due to viruses or resistant bacteria, this also results in the increased
resistance among bacteria including Salmonella typhi and Salmonella
This study provided much needed information and alarms us to the increasing
prevalence of multi-antibiotic resistant Salmonella typhi and Salmonella
paratyphi causing blood stream infections in Bangladesh. The rise in antibiotic
resistance in blood isolates emphasizes the importance of sound hospital infection
control, rational prescribing policies and the need for new antimicrobial drugs
and vaccines. Our results seem helpful in providing useful guidelines for choosing
an effective antibiotic in cases of septicaemia. Specific antibiotic utilization
strategies like antibiotic restriction, combination therapy and antibiotic recycling
may help to decrease or prevent the emergence of resistance.