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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.

Key words: Infection, shigella, salmonella, diarrhea, drug resistance.

INTRODUCTION

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: seyoumbe07@gmail.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 Study design
The study was conducted in two of the four referral hospitals located in Harar, eastern Ethiopia. A convenience sample of Quality control
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. Data analysis
Tables and percentages were used to the describe findings. Cross Specimen collection
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
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 DISCUSSION
(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 Conclusion
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 ACKNOWLEDGMENT
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 REFERENCES
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