Journal of Infectious Diseases and Immunity Vol. 3(8), pp. 134-139, August, 2011 Available online at http://www.academicjournals.org/JIDI ISSN 2141-2375 2011 Academic Journals Full Length Research Paper Antibiotic susceptibility patterns of Salmonella and Shigella isolates in Harar, Eastern Ethiopia Ayalu A. Reda1, Berhanu Seyoum2*, Jemal Yimam2 ,Gizachew Andualem2, Sisay Fiseha2 and Jean-Michel Vandeweerd3
1Department of Public Health, Faculty of Health Sciences, Haramaya University, P. O. Box 235, Harar, Ethiopia.
2Department of Medical Laboratory Technology, Faculty of Health Sciences, Haramaya University, P. O. Box 892, Harar,
3University of Namur, FUNDP, Rue de Bruxelles, Namur, Belgium.
Antimicrobial resistance of Salmonella and Shigella are emerging global challenges. There are no studies conducted on the antimicrobial susceptibility patterns of Salmonella and Shigella in eastern Ethiopia. This study aims to investigate antimicrobial susceptibility patterns of these microbes. A total of 244 diarrheic stool specimens were collected and cultured for screening of microbes using standard methods. Afterwards, the isolates were confirmed as Salmonella or Shigella by using a battery of biochemical reactions. Antibiotic susceptibility testing was performed using six selected drugs (ampicillin, amoxicillin, tetracycline, chloramphenicol, norfloxacin, and gentamicin). 28 (11.5%) Salmonella and 17 (6.7%) Shigella organisms were isolated from 244 stool samples. Sensitivity of the Salmonella isolates were 0.0% to ampicillin; 0.0% to amoxicillin; 14.2% to tetracycline; 28.6% to chloramphenicol; 89.3% to norfloxacin; and 92.8% to gentamicin. Shigella had sensitivities of 0.0% to ampicillin; 0.0% to amoxicillin; 11.8% to tetracycline; 41.2% to chloramphenicol; 88.2% to norfloxacin; and 94.1% to gentamicin. A high level of antimicrobial resistance was detected in both Salmonella and Shigella isolates. The organisms developed complete resistance to ampicillin and amoxicillin. The results of the study demonstrated that gentamicin and norfloxacin are drugs of choice for treating diarrhea caused by these microbes.
Keywords: Infection, shigella, salmonella, diarrhea, drug resistance.
Salmonella and Shigella infections are major global
cause self-limited gastro-enteritis and the more severe
public health problems. Two hundred million to more than
forms of systemic typhoid fever (Kasper et al., 2005;
one billion cases of diarrhea result worldwide due to non-
Goburn et al., 2007). Shigella species are limited to the
typhoidal Salmonella every year leading to 3 million
intestinal tract of humans and cause bacillary dysentery
deaths (Goburn et al., 2007). Ninety-nine percent of the
leading to watery or bloody diarrhea. They are
200 million cases and more than 650,000 deaths per year
transmitted through ingestion of contaminated food and
that result from infection with Shigella commonly occur in
water. These infections are prevalent in developing
developing countries, primarily among children and
countries where lack of clean water supply, lack of proper
young adults (Kasper et al., 2005; Niyogi. 2005; Khatun
sewage disposal system and flies aggravate the spread
et al., 2011). Salmonella and Shigella cause mild to
of the diseases (Kasper et al., 2005). Epidemiological
severe forms of intestinal tract infection. Salmonel a
surveillance is an essential component in controlling
Salmonella and Shigella infections (Sharma et al., 2005).
Drug resistance is the decreased sensitivity or the
complete insensitivity of microbes to drugs that cause cell
*Corresponding author. E-mail: email@example.com.
death or inhibition of growth (Sharma et al., 2005). Anti-
microbial resistance of Salmonella and Shigella are
transported to the laboratory using Cary Blair transport media for
emerging global challenges, especially in developing
further processing and analysis (National Committee for Clinical
countries where there is an increased misuse of
Laboratory Standards, 2004). Using sterile swabs, the samples were directly inoculated onto plates of deoxycholate citrate (DCA)
antimicrobial agents in humans and animals (Kasper et
agar (Oxoid CM 35; Oxoid Ltd, UK) and xylose lysine deoxycholate
al., 2005). In most developing countries, laboratory
(XLD) agar (Oxoid CM 469; Oxoid Ltd, UK) and the plates were
investigations of Shigella and Salmonella are diagnostic
incubated aerobically at 37°C for 24 h.
challenges due to lack of adequate facilities that enable
The same samples were then plated onto Selenite F broth (Mast
culture and antimicrobial susceptibility testing (Collee et
Diagnostics DM 210, Mast Diagnostics, UK) and incubated as aforestated for enrichment. Following the incubation of Selenite F
al., 1999). As a result, there is limited awareness of the
broth, a loopful was streaked onto both DCA and XLD pates and
prevalence of infections and antimicrobial resistance
incubated at 37°C for 24 h. One to three colonies suspected to be
(Cook et al., 2003; Sharma et al., 2005). Also, the
Salmonella or Shigella (small red colonies on XLD and colorless
injudicious use of antibiotics by patients and physicians
colonies sometimes with black spot on DCA) were selected, purified
alike in many developing countries such as Ethiopia has
by streaking on to nutrient agar plates and characterized
lead to an increased antibiotic resistance and in turn
biochemically using Klingler Iron Agar (KIA), Urease tests, motility and indole test (Biomerieux, France) (National Committee for
reduced therapeutic efficacy in these countries (Okeke et
al., 2007; Asrat, 2008). Examining the antibiotic
susceptibility patterns of pathogens is important toward
tailoring treatment to the ever changing resistance
Antimicrobial susceptibility testing
patterns and distribution of pathogenic bacteria. In
Ethiopia, Salmonella and Shigella have been reported to
The disc diffusion was performed to test susceptibility of Salmonella and Shigella isolates using standard procedures (Bauer et al.,
be resistant to first line antibiotics such as ampicillin,
1966). In brief, a MacFarland 0.5 standardized suspension of the
tetracycline and chloramphenicol (Assefa et al., 1997;
bacteria in 0.8% sterile saline was prepared and swabbed over the
Mache et al., 1997; Roma et al., 2000; Mache, 2002;
entire surface of Mueller Hinton agar (Oxoid) with a sterile cotton
Yismaw et al., 2006; Asrat, 2008; Tiruneh, 2009; Beyene
swab. A ring of disks of each (Mast Diagnostics, UK) containing
et al., 2011). To date, there are no studies examining the
single concentrations of each antimicrobial agent was then placed
prevalence and antibiotic susceptibility patterns of
onto the inoculated surface. After overnight incubation at 37°C, clear zones produced by antimicrobial inhibition of bacterial growth
Salmonella and Shigella pathogens in eastern Ethiopia.
were measured in mm using a straight line ruler. The diameter of
Therefore, the aim of this study was to examine antibiotic
the zone was read using an interpreting chart for zone sizes
susceptibility of these pathogens among diarrheal
(NCCLS, 1998). For the susceptibility testing, the following six
patients in the city of Harar in eastern Ethiopia.
antimicrobial drugs and concentrations were used: ampicillin 10 µg,
chloramphenicol 30 µg, gentamycin 30 µg, norfloxacin 10 µg,
amoxicillin 20 µg and tetracycline 30 µg (Oxoid Ltd, UK). Findings
MATERIALS AND METHODS
of antibiotic resistance testing were recorded as susceptible,
intermediate, and resistant (National Committee for Clinical
The study was conducted in two of the four referral hospitals
located in Harar, eastern Ethiopia. A convenience sample of
patients who visited the adult outpatient departments of Hiwot Fana
and Misrak Arbegnoch teaching hospitals from January 2007 to
Quality control was set up using an Escherichia coli strain (ATCC
February 2007 for complaints of diarrheal disease was included in
25922) which was susceptible to all the tested drugs (ampicillin, 10
the study. These hospitals were selected for the study because of
µg; chloramphenicol, 30 µg; gentamycin, 30 µg; norfloxacin, 10 µg;
their close affiliation to the Faculty of Health Sciences of Haramaya
amoxicillin, 20 µg; and tetracycline, 30 µg).
University where the laboratory used for this study is located. The
patient flow of the hospitals ranges from rural and urban dwellers to
clients of diverse socioeconomic and ethnic backgrounds.
Tables and percentages were used to the describe findings. Cross
tabulations were used to examine the levels of drug resistance to
the respective antibiotics. Data analysis was performed with SPSS
244 stool specimens were collected from all patients that presented
with diarrhea (defined three or more loose stools per day) using dry, clean, leak proof, and wide mouth stool containers. Participants
who took antibiotics for the diarrheal attack were excluded from the
Ethical clearance was obtained from the Research Ethics
Isolation and identification of bacteria
Committee of the Faculty of Health Sciences of Haramaya
University. Informed consent was requested from patients.
For detection of Salmonella and Shigella isolates, about 1 to 2 g of
Anonymous forms were used to collect data required for the study
feces was collected. Subsequently, the specimens were
There seems to be complete resistance to ampicillin
In the study period, a total of 244 diarrheal stool samples
and amoxicillin by Salmonella organisms in the study
were collected for culture and antibiotic susceptibility
which is in disagreement with reports from other parts of
testing. Twenty-eight (11.5%) Salmonella and 17 (6.7%)
the country (Mache et al., 1997; Asrat, 2008). This is a
Shigella were isolated. The mean age of the patients
sharp increase from earlier reports indicating the
from whom either Salmonella or Shigella microbes were
aggravating problem of drug resistance by these
isolated was 26 years (SD ±15.4; Range = 11 months to
microbes over the years. Unlike our findings, a high level
70 years). There was one infant of 11 months with a
of resistance to gentamicin (75.6%) was reported from
Shigella infection. Twenty-two (48.9%) were males while
Gondar, northwest Ethiopia by Asrat (2008), whereas the
23 were (51.1%) females. Nine incomplete or empty
same author reports absence of resistance to norfloxacin.
forms were discarded. The most common appearance of
In rural western Kenya, Brooks et al. (2006) found a
diarrhea was mucoid for both Salmonella (12, 42.8%) and
lower level of resistance to the antibiotics used in this
Shigella (9, 52.9%) as displayed in Table 1. The highest
study as well as elsewhere in the country (Ashenafi et al.,
level of resistance was detected for ampicillin and
1985; Mache et al., 1997; Asrat, 2008). The only
amoxicillin in which all (100%) Salmonella and Shigella
exception was gentamicin to which 13.0% of Salmonella
isolates were found to be resistant. The highest level of
isolates in Kenya were resistant (Brooks et al., 2006).
Compared to studies reported in other parts of the
norfloxacin, where, respectively, 92.8 (26) and 89.3%
country (Assefa et al., 1997; Mache et al., 1997; Roma et
(25); and 94.1% (16) and 88.2% (15) of the Salmonella
al., 2000; Yismaw et al., 2006; Asrat, 2008; Tiruneh,
and Shigella isolates were susceptible (Table 2).
2009), Shigella isolates had a lower level of resistance to
chloramphenicol (29.4%). We detected a high level of
susceptibility to gentamicin. Excepting reports by Tiruneh
(2009) and Gedebou et al. (1982) from Gondar who
reported resistance levels of 12.2 and 25% respectively.
Our findings show that except for gentamicin and
Shigella organisms in this study seem to be highly
norfloxacin, the organisms in this study have a high level
susceptible to gentamicin. This is similar to reports from
of resistance to ampicillin, amoxicillin, tetracycline and
other parts of the country (Ashenafi et al., 1985; Assefa
chloramphenicol. In Tables 3 and 4, the findings of this
et al., 1997; Roma et al., 2000) including a report from
study are displayed against findings from other parts of
Kenya (Brooks et al., 2006). Unlike studies in other
the country and neighboring Kenya (Brooks et al., 2006)
places (Assefa et al., 1997; Mache et al., 1997; Roma et
and Eritrea (Naik, 2006). The fact that diarrheal patients
al., 2000; Yismaw et al., 2006; Asrat, 2008; Tiruneh,
with Salmonellosis or Shigellosis present with mucoid
2009) including neighboring Eritrea (Naik, 2006) and
and bloody diarrhea is an interesting finding that may
Kenya (Brooks et al., 2006), Shigella isolates are
have an impact on the way practitioners treat diarrhea in
completely resistant to ampicillin and amoxicillin.
the study area. It is reported (Roma et al., 2000) that
However, there seems to be a similar pattern of high
physicians in Ethiopia wrongly treat bloody diarrhea as an
resistance to these drugs in the studies in the rest of the
amoebiasis attack. However, our findings are in sharp
country, even though lower in extent than our findings
contrast to a study in Addis Ababa by Asrat who reported
(Table 4). This could be due to the fact that ampicillin and
that in addition to mucoid (8.4%), and bloody (6.8%)
amoxicillin have been used in the country for a long time
diarrhea, the majority (82.4%) of the diarrhea samples in
and because of their easy availability and potential for
which Salmonella and Shigella were isolated, had a
misuse. Even though a reduced level of resistance was
watery nature (Asrat, 2008). This may reflect underlying
detected for tetracycline (70.6%), compared to ampicillin
variations in strain patterns from place to place. In
and amoxicillin, a relatively similar pattern of resistance
developing countries (Kasper et al., 2005) Shigella
(74 to 97.3%) was reported from other parts of the
dysenteriae and Shigella flexneri are prevalent species
country (Assefa et al., 1997; Roma et al., 2000; Yismaw
causing mucoid to bloody diarrhea, while in the United
et al., 2006; Asrat, 2008) and outside (Karuiki et al.,
States where Shigella sonei and Shigella boydi species
2001). The organisms seem to also have increased their
predominate, Shigellosis is commonly associated with
resistance to the drugs from lower levels reported earlier
mild watery diarrhea. While S. dysentriae are consistently
(Gedebou et al., 1982; Mache et al., 1997) to levels of
associated with dysentery, it is less common for S.
more than 90% in reports by Asrat (2008) and Truneh et
flexneri to cause bloody diarrhea (Kasper et al., 2005).
al. (Truneh. 2009) in 2009. This is similar to the pattern
The fact that S. flexneri made up 54% of the isolates in
across the globe where the organisms are consistently
Asrat (Asrat, 2008) may explain the watery diarrhea in his
increasing their resistance to these commonly used first
sample. These findings may indicate the need for strain
line drugs (Karuiki et al., 2001; Sharma et al., 2005;
identification in our study area, in order to better
Okeke et al., 2007). Resistance is a natural biological
understand the prevalent strains and their clinical
response of microbes to antimicrobials and is currently a
Table 1. Appearance of diarrhea in patients with Salmonella and Shigella infection in Harar, eastern Ethiopia, between January to February 2007. Organism isolated Appearance of diarrhea Salmonella (n, %) Shigella (n, %) Table 2. Antibiotic susceptibility of Shigella and Salmonella isolates among diarrheic patients in Harar, eastern Ethiopia, between January to February 2007. Outcome of susceptibility test Organism isolated Susceptible (n, %) Intermediate (n, %) Resistant (n, %) Salmonella (N=28) Shigella (N=17) Table 3. Comparison of antimicrobial resistance by Salmonella isolates in the current study with reports from the previous literatures. Rural western, Addis Ababa, Ethiopia Gondar, Ethiopia This study Kenya Brooks et al., Gondar, Ethiopia Antibiotics Ashenafi et al., 1985 Mache et al., 1997 2001 (Brooks et al., Harar, Ethiopia (Asrat, 2008) (Ashenafi et al., 1985) (Mache et al., 1997)
worrisome scenario affecting many parts of the world
resistance by pathogens causing gastroenteritis in the
(Sharma et al., 2005; Khatun et al., 2011). Apart from
setting of a developing country like Ethiopia. These
intrinsic resistance, gene transfer and mutation are
include frequent overuse, misuse and factors related to
among the underlying mechanisms involved in the
the potency and quality of antimicrobials and the
development of antimicrobial resistance by microbes
distribution of resistant strains (Sharma et al., 2005;
(Sharma et al., 2005). Several factors contribute to
Asrat, 2008). In addition, syndromic diagnosis and
Table 4. Comparison of antimicrobial resistance by Shigella isolates in this study with reports from the previous literatures. Addis Ababa, Addis Ababa, Rural western Ethiopia Gedebou Ethiopia Ethiopia Ethiopia Ethiopia Kenya Brooks Ethiopia Eritrea Naik, Ethiopia Ethiopia Ethiopia Antibiotics et al., 1982 Ashenafi et al., Assefa et al., Mache et al., Roma et al., et al., 2001 Yismaw et al., 2006 (Naik, This study Asrat, 2008 Tiruneh, 2008 (Gedebou et al., 1985 (Ashenafi 1997 (Assefa 1997 (Mache 1998 (Roma (Brooks et al., 2005 (Yismaw (Asrat, 2008) (Tiruneh, 2009) et al., 1985) et al., 1997) et al., 1997) et al., 2000) et al., 2006)
diagnostic imprecision usually force physicians to
multiple antibiotics which would have provided us
opt for broad spectrum antibiotics such as
with further insight into the distribution of strains
amoxicillin and tetracycline, over prescribing; and
and the extent of antibiotic susceptibility patterns
In conclusion, except for gentamicin and
less antibiotic diversity which lead to the
in the area. However, this study is a pragmatic
norfloxacin for which both Salmonella and
emergence and spread of antimicrobial resistance
one, given that in the study area in particular, and
Shigella isolates were highly susceptible, a high
(Okeke et al., 2007). For instance, since non-
in Ethiopia in general, antibiotics are prescribed
level of antimicrobial resistance was detected.
typhoidal gastro-enteritis is usually self-resolving,
on empirical bases without implementing the
Notably, the organisms seem to have developed
antibiotic treatment is not commonly recom-
commonly recommended strain isolation and
complete resistance to ampicillin and amoxicillin.
mended (Kasper et al., 2005; Okeke et al., 2007).
susceptibility testing procedures (Kasper et al.,
We assert that gentamicin and norfloxacin may be
However, physicians in our study area are
2005). The small sample size of the current study
drugs of choice for treating diarrheal attacks by
observed to prescribe medication on an empirical
may also be a limitation. However, given the lack
these organisms in the study area. It is
basis (Yismaw et al., 2006). Fortunately, there
of previous studies in the area, it could provide
recommended that a more rigorous study of the
seems to be limited resistance to the drugs
valuable information to clinicians and researchers.
prevalence, antimicrobial susceptibility pattern
gentamicine and norfloxacine. However, given
As the study is an in vitro one, it may not
and underlying mechanisms of drug resistance by
current trends in the country, unless concerted
necessarily reflect in vivo resistance patterns and
Salmonella and Shigella isolates be conducted. In
efforts are made to stem the unrestricted use of
patient outcomes. Unlike previous studies in other
addition, treatment needs to be based on species
antimicrobials in the area, it will not probably be
parts of the country that assessed Salmonella or
identification and susceptibility testing rather than
long before the microbes develop resistance to
Shigella only (Gedebou et al., 1982; Mache et al.,
the currently practiced empirical treatment.
these expensive drugs and complicate effective
1997; Mache et al., 1997; Roma et al., 2000;
Yismaw et al., 2006; Tiruneh, 2009), the fact that
One of the limitations of this study was that due
the current study examined antimicrobial suscep-
to lack of facilities, it was not possible to conduct
tibility tests for both Salmonella and Shigella is
We thank Haramay University for funding the
identification of strain groups and susceptibility to
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Naturopathic Considerations in the Treatment of Postpartum Depression Respectfully Submitted by Trang Q. Duong, N.D. The “postpartum blues” or “baby blues” is a transient state of tearfulness, anxiety, irritation, and restlessness associated with hormonal changes (a steep drop in estrogen and progesterone) that occur in the first few days after birth. Some 50-80% of women in Western cultur
Finite groups and their characters (Paper B2b)Hilary Term 2007: Sheet 0: Specimen answersRemind yourself about Lagrange’s Theorem and its proof. Show that Alt (4) has no subgroup of order 6. See any textbook of algebra or group theory. Indeed, it is a good idea to see several and comparetheir treatments of Lagrange’s Theorem. Let G := Alt (4) and suppose that G had a subgroup H of order